Dodecanol-Modified Petroleum Hydrocarbon Degrading Bacteria for

Dec 2, 2015 - Effective emulsification of oil in seawater plays an important role in the marine oil spill remediation process. The negative effects of...
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Dodecanol-Modified Petroleum Hydrocarbon Degrading Bacteria for Oil Spill Remediation: Double Effect on Dispersion and Degradation Haiyue Gong, Mutai Bao, Guilu Pi, Yiming Li,* Aiqin Wang, and Zhining Wang Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao 266100, Shandong Province, China S Supporting Information *

ABSTRACT: Effective emulsification of oil in seawater plays an important role in the marine oil spill remediation process. The negative effects of chemical surfactants have necessitated a search for alternative dispersant that are sustainable and environmentally friendly. After modifying with dodecanol, petroleum hydrocarbon degrading bacteria, Bacillus cereus S-1, was elucidated to produce an extremely stable oil-in-water (o/w) Pickering emulsion, just liking a solid particle emulsifier. In an appropriate concentration range, the presence of dodecanol improved the surface hydrophobicity and wettability of bacterial cells, which was responsible for their adsorption at the oil−water interface. When a sufficient amount of bacteria was added, only a small amount of dodecanol was required to obtain stable emulsions. However, stable emulsion was not prepared with unmodified Bacillus cereus S-1 cells. Scanning electron microscopy (SEM) and confocal laser scanning microscope (CLSM) images indicated that the effective emulsification was attributed to the formation of a dense bacterial interfacial film around oil drops, providing a steric barrier to impeding droplet coalescence. For application in emulsification of crude oil in seawater, the dodecanol-modified bacterial cells (DMB) were still very effective. In addition to emulsification, DMB remarkably facilitated the oil biodegradation compared to bacterial cells alone. By combining the emulsification and biodegradation of sustainable hydrocarbon degrading bacteria, this work showed a novel strategy for developing an alternative, environmentally friendly remediation technology for marine oil spills. KEYWORDS: Petroleum hydrocarbon degrading bacteria, Dodecanol, Oil emulsification, Oil spill dispersion, Biodegradation

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INTRODUCTION In the past few years, frequently occurring oil spill accidents result in a large amount of petroleum hydrocarbon leaked into the ocean.1 These hazardous contaminants have disastrous consequences for the marine environment. A lot of methods, including physical collection, chemical dispersion, and biodegradation, are used to remove these toxic contaminants. Except for the physical removal method, chemical dispersion and enhanced biodegradation are also important ways in dealing with marine oil spill pollution.2,3 The dispersion of an oil slick is regarded as an emulsification process. An important benefit for dispersion is its capability to stimulate the natural biodegradation process by providing more area available for oildegrading microorganisms.2 After the first priority of physical collection, any oil that is not collected only can be removed by biodegradation. So, stimulating interest in this process is a © 2015 American Chemical Society

promising option. While chemical surfactants are effective for marine oil spill dispersion, their toxicity is still a major concern of some researchers.4 Therefore, nontoxic, environment friendly, and effective dispersants would be potential candidates in the treatment of oil spills.5,6 It is well known that when oil is dispersed in water the oil− water interfaces can be stabilized by colloidal particles. Such emulsions are known as Pickering emulsions.7 Pickering emulsions are widely applied in a number of industrial processes, especially in the industries needing to avoid the use of surfactant. The mechanism of emulsion stabilized by particles is attributed to the adsorption of particles at the oil− Received: August 25, 2015 Revised: October 9, 2015 Published: December 2, 2015 169

DOI: 10.1021/acssuschemeng.5b00935 ACS Sustainable Chem. Eng. 2016, 4, 169−176

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alone was not an effective emulsifier due to its strong hydrophilicity. However, DMB can be adsorbed to the oil− water interface as an emulsifier, as a consequence of the enhanced hydrophobicity of the Bacillus cereus S-1 surface. To the best of our knowledge, it has never been reported that petroleum hydrocarbon-degrading bacteria treated with dodecanol is used in stabilizing oil-in-seawater emulsions, together with its enhanced biodegradation ability for oil. In the present work, a model oil, n-tetradecane, was used to investigate emulsion formation and the emulsification mechanism first, and then, these results were used to direct the emulsification of crude oil in seawater. Here, we found that DMB was able to stabilize crude oil-in-seawater emulsion effectively, while providing enhanced oil biodegradation.

water interface in the form of a rigid layer, consequently impeding the creaming and coalescence of oil droplets by providing a steric barrier.8,9 The emulsion type, either oil-inwater (o/w) or water-in-oil (w/o), could be obtained based on the wettability of the particles.10 If the three-phase contact angle of the particle is smaller than 90°, it will favor the formation of o/w Pickering emulsions.11 Conversely, a w/o Pickering emulsion is formed when the contact angle is higher than 90°. Many types of colloidal particles have been demonstrated to be an effective Pickering emulsion stabilizer. Examples include clay particles, silica, polymeric particles, and microgel particles.12−15 However, the specific application of these colloid particles to oil spill dispersion is ignored for a long time, comparing with the application of Pickering emulsion in other fields. Recently, Saha et al. found that carbon black particles are effective marine oil spill dispersants because of their strong adsorption ability and peculiar interfacial elasticity at the oil−water interface.16−18 Natural montmorillonite particles promoted the formation of stable dodecane-inseawater emulsion.19 Recently, an important research effort is focused on preparing emulsion stabilized by biobased material because of their environmentally benign nature.20,21 In addition to the colloidal particles mentioned above, living microorganisms can also be used as effective particle emulsifiers. Bacterial cells may be considered as colloid particles because of their size, shape, and composition of cell membrane surface.22 Though some bacteria may produce emulsifiers in the process of metabolism, it is worth noting that washed bacterial suspension without metabolites could emulsify hydrocarbons alone.23−25 Rather than the specific interaction of bacteria with substrate, the bacterial cells with appropriate wettability could attach to the oil−water interface via hydrophobic interactions, just like colloidal particles. For hydrophilic bacterial cells, various methods were used to improve the hydrophobicity of bacterial cells to make them effective emulsifiers.26 In a pioneering work, Dorobantu and coauthors demonstrated that through adsorption at the interface and inhibition of droplet coalescence intact bacterial cells were effective emulsifiers without changing the interfacial tension.23 After modification of the bacterial cell surface by chitosan through electrostatic attraction, a hydrophilic bacteria was proven to promote formation of a Pickering emulsion with high stability.24,25 The unique cell surface characteristic of a hydrophilic Gram-positive bacterium resulted in microbial adhesion at the oil−water interface, thereby a stable o/w emulsion.27 Stable w/o emulsions also can be formed when the bacterial cells are dispersed in organic solvents, which had a potential to be used in bioconversion fields.28 Recently, we found that a microbial consortium isolated from oilfield polluted sludge and a chitosan-modified oil degrading bacteria were capable of emulsifying petroleum hydrocarbons effectively.29,30 So, the use of sustainable microorganisms in oil spill remediation is a promising, environmentally friendly, and cheaper research approach in this field.31 In this paper, a hydrocarbon degrading bacteria, Bacillus cereus S-1, modified with dodecanol was investigated systematically as an emulsifier in forming oil-in-water emulsions. Comparing with inert particles, a dodecanol-modified Bacillus cereus S-1 (DMB) strain may not only act as an effective emulsifier but also have the biodegradation for oil simultaneously. These properties reflect the possibility of a double effect on emulsification and biodegradation, which is very desirable for oil spill remediation. The Bacillus cereus S-1 strain

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EXPERIMENTAL SECTION

Materials. The following reagents were purchased and used as received: dodecanol and n-tetradecane (Aladdin, China), 2.5% glutaraldehyde in sodium phosphate buffer solution (PBS) (Rongping Shanghai Biological Technology Co. Ltd., China), 2,20-azobis(2,4dimethyl valeronitrile) (V65) (TCI Shanghai, China), and nile red and 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). Styrene (Sinopharm Chemical Reagent Co., Ltd., China) was purified before using. Crude oil was supplied by China National Offshore Oil Corporation. In order to protect bacteria from rupture and dehydration in pure water, mineral salts medium (MSM) was used to replace pure water to prepare emulsions. Then, these results were used to guide the preparation of crude oil-in-seawater emulsion. MSM consisted of (L−1): KH2PO4, 3.0 g; NaH2PO4, 3.0 g; (NH4)2SO4, 5.0 g; MgSO4, 0.5 g; NaCl, 5.0 g; CaCl2, 0.02 g. The artificial seawater (ASW) consisted of (L−1): NaCl, 26.726 g; MgSO4·7H2O, 6.67 g; CaCl2, 1.153 g; NaHCO3, 0.2 g; KCl, 0.721 g; MgCl2·6H2O, 4.89 g. The pH of ASW was 7.8. Bacteria and Growth Conditions. A hydrocarbon degrading bacteria, Bacillus cereus S-1, was isolated from seafloor sediment polluted by crude oil, as described in our previous studies.30 The microbial consortium was grown in a beef extract peptone medium shaking at 30 °C with 120 rpm. Bacterial cells were centrifuged at 7046g for 5 min, washed twice with 0.85% NaCl solution, and resuspended in PBS solution to give an optical density of 2.0 at 600 nm (final cell concentration is 6.3 × 108 cells mL−1). Preparation of Emulsion. By mixing oil and a given amount of DMB suspension in MSM, emulsions were prepared in a total volume of 8 mL. The mixture was vigorously shaken by hand first and then stirred at 6000 rpm for 5 min using an IKAT10 homogenizer instrument. Emulsion formation and stabilization was analyzed by measuring the emulsion droplet size and emulsification index (percentage of the height of emulsion layer divided by the total height of the mixture over different days), as well as their time dependence. Toxicity Measurements of Dodecanol. Over a limited range of dodecanol concentration (1.5 g L−1), the emulsion formed in MSM solution was stable in a month, as shown in Figure 4f and h, although these bacterial cells were fragile. The EI value and oil droplet size changed little over time in a month. This was likely because of the enhanced hydrophobicity of the cell surface and the formation of a bacterial interfacial film around the droplet. The combination of these two factors allowed for fine emulsions and a steric barrier, which inhibited the coalescence of oil droplets. In addition, the high affinity between bacteria cells also aided the formation of a bacterial film around the oil droplet.23 Figure 5a showed the effect of dodecanol concentration on the EI value and initial oil droplet size for n-tetradecane emulsions stabilized by DMB. Smaller oil droplet size was attributed to the enhanced wettability and hydrophobicity of bacterial cells in the presence of dodecanol. With an increase in dodecanol concentration, the hydrophobicity of DMB cells was enhanced, which was favorable for their adsorption at the oil− water interface. However, exposure to higher dodecanol concentration disrupted the bacterial inner cytomembrane and lysed the bacteria. Hence, there was an optimal value of dodecanol concentration for emulsification, as shown in Figure 5a. When preparing an oil-in-water emulsion, some dodecanol molecules may dissolve in n-tetradecane because of its oilsoluble properties. So, the concentration of dodecanol that really interacted with bacteria was less than the addition in the emulsion preparation. This explained that the optimal concentration of dodecanol in emulsification was higher than that in the contact angle measurements. It was important to mention that the formation of a stable emulsion utilized only a bit concentration of dodecanol, and the emulsion stability was mainly influenced by the bacteria concentration, as shown in Figure 5b. In the case of a small amount of bacterial cell particles, only a small interfacial area can be stabilized, so large droplets were observed. A large amount of bacterial cells allowed the preparation of very fine emulsions.36 This implied that more bacterial particles would act as stabilizers in the system. So the droplet size of emulsion was decreased, and EI value was enhanced with the concentration of bacteria increasing. However, an efficient emulsification process is required. The emulsification process needs to be powerful enough to create the full interfacial area that the bacterial particles can cover. When the bacterial concentration was higher than 8 × 108 cells mL−1, EI and droplet size changed little.

Figure 6. Fluorescence images of n-tetradecane-in-water emulsion stabilized by DAPI-stained bacterial cells without (a) and with 2.0 g L−1 dodecanol (b) modification. n-Tetradecane was stained with nile red. SEM images of styrene-in-water emulsion stabilized by Bacillus cereus S-1 cells (c, d) and DMB (e, f) (CS‑1= 6.3 × 108 cells mL−1, Cdodecanol = 2.0 g L−1).

that without dodecanol, hydrophilic bacterial cells cannot effectively adsorb at the oil droplet surface. So, phase separation occurred in a few days (Figure S1a). However, a clear bacterial film surrounding an oil droplet was observed when DMB was used as a stabilizer, as shown in Figure 6b. The blue bright fluorescence of DAPI at the edge of the emulsion droplet and the red fluorescence of nile red inside confirmed the adsorption of DMB cells at the oil droplet surface. 173

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Crude Oil Biodegradation by DMB. The effect of Bacillus cereus S-1 and DMB on the biodegradation of crude oil was determined by ultraviolet spectrophotometer. Figure 8a shows

The emulsion droplet was also visualized by SEM. Figure 6c and d show that when unmodified bacterial cells were used as emulsifiers, the oil droplet surface was only covered by a small amount of bacterial cells, which resulted in an unstable emulsion. Whereas, for emulsion stabilized by DMB, the oil droplets were covered tightly by a large amount of agminated DMB cells (Figure 6e, f). These tightly adsorbed DMB cells formed a dense bacterial film at the oil droplet surface. From comparison of these two systems, it can be concluded that dodecanol induced more bacterial cells adsorbed at the oil− water interface. By hydrophobic interactions, bacterial cells were adsorbed and held together at the oil−water interface, which resulted in the formation of a bacterial film. This bacterial film provided stability for the emulsion by resisting the coalescence and deformation of droplets.36 In addition, the affinity between hydrophobic bacterial cells was proved to be a favorable contribution to the self-assembly of cells at the interface and emulsion stabilization.23 The coalescence between oil droplets was prevented by the mechanical barrier of a bacterial film. The mechanical strength of this bacterial film came from aggregation of bacterial particles at the droplet surface. The aggregation of bacterial cells induced by dodecanol was also observed in aqueous phase, as shown in Figure 6b. Crude Oil-in-ASW Emulsions Stabilized by DMB. The effective emulsification of DMB for n-tetradecane in water indicated that DMB had potential applications in crude oil emulsification in the treatment of oil spills. To that end, crude oil-in-ASW emulsions stabilized by unmodified bacterial cells and DMB were prepared, respectively (Figure 7). When DMB

Figure 8. (a) Degradability of crude oil by DMB in different dodecanol concentrations after 6 days. (b) Degradability of crude oil by bacteria and DMB (Cdodecanol = 0.8 g L−1). Inoculation quantity of bacteria suspension (CS‑1 = 6.3 × 108 cells mL−1) is 5%.

the biodegradation of crude oil by bacterial cells modified with different concentrations of dodecanol. The degradation rate of crude oil also showed an optimum value when dodecanol increased, which illustrated a similar trend with the influence of dodecanol on emulsification. When the dodecanol concentration was higher than 1.4 g L−1, it had a negative effect on the biodegradation of crude oil by bacteria. On one hand, the higher concentration of dodecanol resulted in a more hydrophobic bacterial cell, which promoted biodegradation because of more bacterial cells adsorbed at the crude oil−water surface. On the other hand, more dodecanol molecules led to cell lysis, which decreased the biodegradation of oil by bacteria. The natural degradation of crude oil in ASW is also called physical and chemical degradation, which is caused by some physical−chemical factors. This natural degradation is often used as a control. We found lower natural degradation of crude oil but observed a significant increased degradation rate when Bacillus cereus S-1 was present. As shown in Figure 8b, 30% of crude oil was degraded after 14 days. It was worth noting that in the presence of DMB even 45% of crude oil was degraded after 14 days, which showed the further enhanced degradation rate. The remarkable enhanced degradation rate by DMB was because of the increased oil−water interfacial area and enhanced cell surface hydrophobicity. Through effective emulsification of DMB, the oil was dispersed into small droplets, which subsequently resulted in more bacteria coming into direct contact with oil. Kaczorek et al. found that bacterial biodegradation of diesel oil was enhanced upon addition of rhamnolipid or Triton X-100 due to their emulsification.39 In this study, dodecanol did not independently work as an emulsifier and also had no contribution to the degradability of oil (Figure 8a). This indicated that in the process of crude oil biodegradation, the only function of dodecanol was modifying the hydrophobic characteristic of the bacterial cell surface.

Figure 7. Light microscopy images of crude oil-in-ASW emulsions stabilized by unmodified bacterial cells (a) and DMB (b). Emulsions were prepared with 6.3 × 108 cells mL−1 Bacillus cereus S-1 and DMB (0.7 g L−1 dodecanol was used) at a 12.5 vol % crude oil fraction.

was used as an emulsifier, a stable emulsion was formed, and the average emulsion droplet size was ∼90 μm. After 1 month in our lab, this emulsion did not show evident phase separation, and droplet size increased. Comparing with n-tetradecane-inMSM emulsions, we even obtained smaller oil droplet size and higher emulsion stability for crude oil-in-ASW emulsions. Crude oil contains asphaltenes and resins. Merv Fingas et al. confirmed the chemical stabilization of asphaltenes and resins for w/o emulsions.37 The adsorption of asphaltenes at the oil− water interface is a thermodynamically favored process because of the reduced free energy. Other researchers also demonstrated that resins and asphaltenes are both involved in emulsion stabilization, and the emulsions are more stable when the ratio of asphaltene:resin increases.38 So, it was proposed that the presence of asphaltenes and resins in crude oil enhanced the stabilization of crude oil−in−ASW emulsions stabilized by DMB. Our results provided strong evidence that the DMB suspensions has a potential use in dispersing oil spills.

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CONCLUSIONS This work demonstrated DMB as an effective emulsifier and investigated applications in oil spill remediation. Neither Bacillus cereus S-1 cells nor dodecanol was an effective emulsifier alone. However, DMB cells showed an effective emulsification. The major findings included (1) a simple method to prepare stable emulsions with sustainable and low environmental impact of bacteria particles. The emulsion stability was substantially enhanced because of the enhanced 174

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(7) Aveyard, R.; Binks, B. P.; Clint, J. H. Emulsions stabilized solely by colloidal particles. Adv. Colloid Interface Sci. 2003, 100-102, 503− 546. (8) Binks, B. P.; Horozov, T. S., Eds.; Colloidal Particles at Liquid Interfaces: An Introduction. Colloidal Particles at Liquid Interfaces; Cambridge University Press, 2006; Chapter 1. (9) Kraft, D. J.; de Folter, J. W. J.; Luigjes, B.; Castillo, S. I. R.; Sacanna, S.; Philipse, A. P.; Kegel, W. K. Conditions for equilibrium solid-stabilized emulsions. J. Phys. Chem. B 2010, 114, 10347−10356. (10) Binks, B. P.; Lumsdon, S. O. Influence of particle wettability on the type and stability of surfactant-free emulsions. Langmuir 2000, 16, 8622−8631. (11) Lee, K. Y.; Blaker, J. J.; Murakami, R.; Heng, J. Y.; Bismarck, A. Phase behavior of medium and high internal phase water-in-oil emulsions stabilized solely by hydrophobized bacterial cellulose nanofibrils. Langmuir 2014, 30, 452−460. (12) Binks, B. P.; Desforges, A.; Duff, D. G. Synergistic stabilization of emulsions by a mixture of surface-active nanoparticles and surfactant. Langmuir 2007, 23, 1098−1106. (13) Owoseni, O.; Nyankson, E.; Zhang, Y. H.; Adams, S. J.; He, J. B.; McPherson, G. L.; Bose, A.; Gupta, R. B.; John, V. T. Release of surfactant cargo from interfacially-active halloysite clay nanotubes for oil spill remediation. Langmuir 2014, 30, 13533−13541. (14) Tzoumaki, V. M.; Moschakis, T.; Kiosseoglou, V.; Biliaderis, G. C. Oil-in-water emulsions stabilized by chitin nanocrystal particles. Food Hydrocolloids 2011, 25, 1521−1529. (15) Fujii, S.; Read, E. S.; Binks, B. P.; Armes, S. P. Stimulusresponsive emulsifiers based on nanocomposite microgel particles. Adv. Mater. 2005, 17, 1014−1018. (16) Saha, A.; Nikova, A.; Venkataraman, P.; John, V. T.; Bose, A. Oil emulsification using surface-tunable carbon black particles. ACS Appl. Mater. Interfaces 2013, 5, 3094−3100. (17) Katepalli, H.; John, V. T.; Bose, A. The response of carbon black stabilized oil-in-water emulsions to the addition of surfactant solutions. Langmuir 2013, 29, 6790−6797. (18) Powell, K. C.; Chauhan, A. Interfacial tension and surface elasticity of carbon black (CB) covered oil−water interface. Langmuir 2014, 30, 12287−12296. (19) Dong, J.; Worthen, A. J.; Foster, L. M.; Chen, Y.; Cornell, K. A.; Bryant, S. L.; Johnston, K. P.; Truskett, T. M.; Bielawski, K. P. Modified montmorillonite clay microparticles for stable oil-in-seawater emulsions. ACS Appl. Mater. Interfaces 2014, 6, 11502−11513. (20) Kalashnikova, I.; Bizot, H.; Cathala, B.; Capron, I. New Pickering emulsions stabilized by bacterial cellulose nanocrystals. Langmuir 2011, 27, 7471−7479. (21) Russell, J. T.; Lin, Y.; Böker, A.; Su, L.; Carl, P.; Zettl, H.; He, J.; Sill, K.; Tangirala, R.; Emrick, T.; Littrell, K.; Thiyagarajan, P.; Cookson, D.; Fery, A.; Wang, Q.; Russell, T. P. Self-assembly and cross-linking of bionanoparticles at liquid−liquid interfaces. Angew. Chem., Int. Ed. 2005, 44, 2420−2426. (22) Ubbink, J.; Schär-Zammaretti, P. Colloidal properties and specific interactions of bacterial surfaces. Curr. Opin. Colloid Interface Sci. 2007, 12, 263−270. (23) Dorobantu, L. S.; Yeung, A. K.; Foght, J. M.; Gray, M. R. Stabilization of oil-water emulsions by hydrophobic bacteria. Appl. Environ. Microbiol. 2004, 70, 6333−6336. (24) Wongkongkatep, P.; Manopwisedjaroen, K.; Tiposoth, P.; Archakunakorn, S.; Pongtharangkul, T.; Suphantharika, M.; Wongkongkatep, J.; Honda, K.; Hamachi, I. Bacteria interface Pickering emulsions stabilized by self-assembled bacteria−chitosan network. Langmuir 2012, 28, 5729−5736. (25) Archakunakorn, S.; Charoenrat, N.; Khamsakhon, S.; Pongtharangkul, T.; Wongkongkatep, P.; Suphantharika, M.; Wongkongkatep, J. Emulsification efficiency of adsorbed chitosan for bacterial cells accumulation at the oil−water interface. Bioprocess Biosyst. Eng. 2015, 38, 701−709. (26) Ly, M. H.; Naïtali-Bouchez, M.; Meylheuc, T.; Bellon-Fontaine, M.; Le, T. M.; Belin, J.; Waché, Y. Importance of bacterial surface

cell surface hydrophobicity by dodecanol. This simple modification by dodecanol resulted in more bacterial cells adsorbed at the oil−water interface. The tightly adsorbed DMB cells at the oil−water interface provided a dense bacterial interfacial film, which was responsible for the long-term stabilization of emulsion. (2) We showed evidence of enhanced crude oil biodegradation by DMB in an appropriate dodecanol concentration range. The remarkable enhanced biodegradation was attributed to the enhanced hydrophobicity of bacterial cells and the increased oil−water interfacial area available to the bacteria because of the effective emulsification of DMB. This study demonstrated an efficient remediation method for marine oil spill pollution with sustainable hydrocarbon degrading bacteria. The enhancement of cell surface hydrophobicity promoted the accessibility of bacterial cells to the oil droplet, which resulted in enhanced emulsification and biodegradation. Thus, the strategy of modifying surface characteristics of hydrocarbon-degrading bacteria seems to be a promising method to enhance emulsification and biodegradation simultaneously for oil spill remediation.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.5b00935. Bacteria growth of Bacillus cereus S-1 in different conditions after 24 h; photographs of tetradecane-inwater emulsion stabilized by Bacillus cereus S-1 and dodecanol alone in MSM. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: 86-532-66782509. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research is supported by the Applied Basic Research Programs of Qingdao in China (14-2-4-119-jch), Natural Science Foundation of Shandong Province (ZR2014DQ026), and National Natural Science Foundation of China (41376084). This is MCTL contribution No. 96.

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